Eukaryotic gene expression is governed at multiple levels, involving local modes of regulation with short-range effects and global modes of regulation effective over extended distances. Within the transcriptional unit of a gene, cis-acting regulatory elements, such as promoters and enhancers, are positioned locally to efficiently recruit the transcriptional machinery to the DNA template to enable the transcription of the gene to an RNA product. Eukaryotic genes may be arranged in a linear sequence within chromosomes that are structurally and functionally subdivided into either transcriptionally active or transcriptionally inactive states. Heterochromatin comprises transcriptionally inactive domains consisting of highly condensed DNA/Histone complexes that are insensitive to DNase I treatment. Euchromatin comprises transcriptionally active domains with complexes of DNA/Histones less densely packed than in heterochromatin that are sensitive DNase I treatment.
Transcriptionally active and inactive regions of the genome are structurally distinguishable due to differential post-translational states that result from various modifications of DNA-associated histones by acetylation, methylation, phosphorylation, and ubiquitinylation. These modifications can be essential for tissue-specific regulation of gene expression, especially during differentiation processes. Euchromatin has been shown to correlate with the hyperacetylation of associated histones. In particular, the acetylation of several lysine residues and the methylation of lysine 4 in histone H3 are characteristic features of transcriptionally active genes. Transcriptionally inactive regions, by contrast, are organized into heterochromatic structures that are characterized by deacetylation of H3 and H4, and lysine 9 methylation of H3. The existence of eukaryotic genes within dynamic chromatin states contributes significantly to the phenomenon of position-effect variegation that frequently precludes stable, long-term heterologous gene expression upon random integration of exogenous DNA into a host chromosome. Because a substantial portion of the eukaryotic genome exists in the transcriptionally inactive heterochromatin state, random integration of exogenous genes into a heterochromatin environment likely results in transgene silencing. For example, gene therapy has only been partially successful, because of position-effect variegation that results from random insertion of transgenes into heterochromatin regions, leading to transgene suppression [G. H. Karpin, Curr. Opin. Genet. Dev. 4: 281-291 (1994)].
Unpredictable and low yields in recombinant protein production, which significantly reduces the rate at which therapeutic proteins are brought to the market, have also been attributed to position-effect variegation. Production yields from cultivated mammalian cells, and from transgenic plants and animals, are still not sufficient to meet growing demands. It is estimated that tons of human serum albumin are needed annually, which are currently produced by processing 50,000 liters of human plasma. Several hundred kilograms of proteins, such as collagen, thrombin, and recombinant therapeutic antibodies, are in demand on a global scale, but the demand has not been fulfilled [Florian M. Wurm, Nature Biotechnology 21: p34-35 (2003)]. There is a continuing need for improvements in recombinant technologies, in particular, improvements in vector designs for enabling more robust expression of heterologous genes within a chromatin environment, such as stably integrated exogenous genes within a host chromosome. Expression vectors that can improve the predictability, yield, and stability of protein expression are of significant value to the biotechnology industry and the biomedical community.